A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

• Published in: Analytica chimica acta 2015-08, Vol.887, p.209-215
• Main Authors: Cywiński, Piotr J., Olejko, Lydia, Löhmannsröben, Hans-Gerd
• Format: Article
• Language: English
• Subjects: Aptamer; Aptamers, Nucleotide - chemistry; Carbocyanines - chemistry; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - chemistry; Fluoroassay; FRET; Humans; L-selectin; L-Selectin - analysis; L-Selectin - blood; Lanthanide; Limit of Detection; Luminescence spectroscopy; Luminescent Measurements - methods; Terbium - chemistry
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2015.06.045

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). [Display omitted] •Tb-based FRET assay with aptamers toward a protein is presented for the first time.•L-selectin can be detected in concentrations relevant for the Alzheimer's Disease.•The assay can be realized in one hour with the LOD equal to 10 ng/ml.